Passive Ca2+ fluxes across the membrane of sarcoplasmatic reticulum of skeletal muscles. The effect of calcium channel blockers

It was found that the initial rate of passive KC1-stimulated Ca2+ influx into sarcoplasmic reticulum (SR) vesicles follows the saturation kinetics at Ca2+ concentrations of 8-10 mM. The inhibitory effect of Ca2+ channel blockers (La3+, Mn2+, Co2+, Cd2+, Mg2+) on passive Ca2+ influx into SR vesicles is competitive with respect to Ca2+. These blockers also inhibit the initial fast phase of Ca2+ efflux from Ca2+-loaded SR vesicles. Verapamil (0.1-0.5 mM) added to the incubation mixture has no effect on passive Ca2+ fluxes across the SR vesicle membrane or on Ca2+ binding and ATP-dependent Ca2+ accumulation. However, preincubation of SR vesicles with verapamil (18 hours, 4 degrees C) or its introduction into the medium for SR vesicle isolation leads to the inhibition of passive Ca2+ fluxes.     

Cation selectivity characteristics of the reconstituted voltage-dependent sodium channel purified from rat skeletal muscle sarcolemma

In this report, the alkali metal cation selectivity of the purified, voltage-dependent sodium channel from rat skeletal muscle is described. Isolated sodium channel protein (980-2840 pmol of saxitoxin binding/mg of protein) was reconstituted into egg phosphatidylcholine vesicles, and channels were subsequently activated by either batrachotoxin (5 X 10(-6) M) or veratridine (5 X 10(-4) M). Activation of the reconstituted sodium channel by batrachotoxin permitted rapid specific influx of cations into channel-containing vesicles. Quenched flow kinetic techniques were adapted to allow resolution of the kinetics of cation movement. Uptake rates for 42K+, 86Rb+, and 137Cs+ were measured directly and half-times for equilibration at 18 degrees C were determined to be 350 ms, 2.5 s, and 10 s, respectively, in this vesicle population. 22Na+ equilibration occurred within the mimimum quenching time of the apparatus (90 ms) but an upper limit of 50 ms at 18 degrees C could be assigned to its half-time. Based on this upper estimate for Na+, cation selectivity ratios of the batrachotoxin-activated channel were Na+ (1):K+ (0.14):Rb+ (0.02):Cs+ (0.005). Toxin-stimulated influx could be blocked by saxitoxin with a Ki of approximately 5 X 10(-9) M at 18 degrees C. Rates of cation movement through veratridine-activated channels were much slower, with half-times of 1.0, 1.2, 2.0, and 2.6 min at 36 degrees C for Na+, K+, Rb+, and Cs+, respectively. The temperature dependences of batrachotoxin and veratridine-stimulated cation uptake were markedly different. The activation energies for 86Rb+ and 137Cs+ movement into batrachotoxin-activated vesicles were 7.6 and 6.1 kcal/mol, respectively, while comparable measurements for these two cations in veratridine-activated vesicles yielded activation energies of 31 kcal/mol. Measurements of cation exchange with batrachotoxin-activated channels may reflect characteristics of an open sodium channel while the process of channel opening itself may be rate-limiting when veratridine is used for activation.     

A study of passive Ca2+ flow through the sarcoplasmic reticulum of skeletal muscles. II. Stimulation of passive Ca2+ influx with monovalent cations and anions

The initial rate of passive Ca2+ influx into "heavy" and "light" fractions of sarcoplasmic reticulum (SR) vesicles increases in the presence of univalent cation chlorides. Stimulation of passive Ca2+ influx decreases in the following order: KCl + valinomycin-KSCN- + valinomycin greater than KSI = NaCl greater than choline chloride. K-gluconate + valinomycin and K-gluconate have no effect on the passive Ca2+ influx into SR vesicles. It is supposed that KCl-stimulation of passive Ca2+ influx into SR vesicles under conditions used may be caused by depolarization of the SR membrane.     

Effects of adenine nucleotides on the Ca2+-gated cation channel in sarcoplasmic reticulum vesicles

The effects of nucleotides on the Ca2+-gated cation channel in sarcoplasmic reticulum (SR) vesicles were studied by measuring choline influx. The choline influx was measured by following the change in scattered light intensity using the stopped flow technique. ATP enhanced the Ca2+-induced choline influx. The activation followed a single-site titration curve with a dissociation constant of 1.0 +/- 0.5 mM, independent of the Ca2+ concentration. ATP seems to increase the pore radius or number of channels without affecting the gating mechanism of the Ca2+-gated cation channel. ADP, AMP, and adenine enhanced the choline transport in a manner similar to ATP, but cAMP, ITP, UTP, CTP, and GTP did not. The apparent dissociation constants and the maximal activations were as follows: ATP 1.0 mM, 28-fold; ADP 0.9 mM, 18-fold; AMP 0.6 mM, 7-fold, and adenine 0.4 mM, 4-fold. Adenine and AMP behaved as a competitive inhibitor for the activation by ATP. These results are consistent with the Ca2+-induced Ca2+ release observed in skinned muscle fiber and isolated SR.     

Tetraphenylboron increases choline permeability through a calcium release channel of isolated sarcoplasmic reticulum

The lipophilic anion tetraphenylboron (TPB-) but not the lipophilic cation tetraphenylphosphonium (TPP+) increased the choline permeability of isolated sarcoplasmic reticulum (SR). Choline permeability was mainly measured by the stopped flow method by following the change in scattered light intensity. TPB- and TPP+ did not affect the choline permeabilities of liposomes, liver microsomes, or denatured SR vesicles. These phenomena are similar to the Ca2+ release phenomena activated by TPB- reported by Shoshan, MacLennan, and Wood (J. Biol. Chem. 258, 2837 (1983)). These results strongly suggest that TPB- activates a pre-existing channel of SR membrane and choline permeates through the same channel as that for the Ca2+ release. This channel is different from that for the Ca2+-induced Ca2+ release. The former is present in all of the vesicles formed by fragmented SR, while the latter is rich in the heavy fraction of fragmented SR and poor in the light fraction. The channel specificities for permeable ions are different from each other. For example, the latter passes Tris+ but the former does not. The physiological role of this channel is not clear at present.     

The reconstituted isolated uncoupling protein is a membrane potential driven H+ translocator.

The isolated uncoupling protein (UCP) from brown fat adipose tissue mitochondria has been reconstituted into artificial phospholipid vesicles. Because of the high lability of H+ transport, several new steps have been introduced in the reconstitution; the detergent octyl-POE, the addition of phospholipids to mitochondria prior to solubilization and purification, the vesicle formation by rapid removal of detergent with polystyrene beads and of external salts by a mixed ion exchange. In the K+-loaded proteoliposomes, H+ influx can be induced by a diffusion potential on addition of valinomycin. H+ influx is inhibited to more than 90% by GTP addition, in the assay for UCP activity. By reversing delta psi with external K+, H+ efflux is measured, however, at a four times lower rate. In vesicles loaded with internal GTP, H+ influx is fully inhibited but can be activated by Dowex-OH treatment to an even higher rate than that found in the GTP-free vesicles. Binding studies with GTP show that most of the active UCP are oriented with the binding site outside as in mitochondria, and that in GTP-loaded vesicles GTP is also bound at the outside. The rate of H+ transport is linearly dependent on the membrane potential. Despite the ordered orientation, there is no 'valve' mechanism, since there is H+ efflux with a reversed potential. pH dependency is only small between pH 6.5 and 7.5, indicating that the H+-translocating site differs from the highly pH-dependent nucleotide-binding site. The turnover number of reconstituted UCP is commensurate with mitochondrial function and indicates a carrier instead of a channel-type H+ transport.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of local anesthetics on the passive permeability of sarcoplasmic reticulum vesicles to Ca2+ and Mg2+

Sarcoplasmic reticulum vesicles are used here as model membrane system to question the hypothesis of enhancement of permeability of cations by anesthetics, particularly that of Ca2+ and of Mg2+. The effects of dibucaine (up to 800 microM), tetracaine (up to 2 mM), lidocaine (up to 10 mM) and procaine (up to 10 mM) on the permeability of these membranes to Ca2+ and Mg2+ have been measured. We have used an experimental approach based on the light scattering method (Kometani, T. and Kasai, M. (1978) J. Membrane Biol. 41, 295-308). It has been found that all the local anesthetics cited above markedly increase the permeability of sarcoplasmic reticulum vesicles to Mg2+ and, in the concentration range tested herein, only dibucaine and tetracaine increase the permeability to Ca2+. The kinetic analysis of the time dependence of the light-scattering data after the osmotic shock shows that, in the absence of local anesthetics, the Mg2+ influx can be described as proceeding through a unique type of channel. However, Ca2+ influx appears to involve two channel of different kinetic properties. Because the relative fraction of both types of Ca2+ channel is similar to the average ratio between light and heavy vesicles in unfractionated sarcoplasmic reticulum, we suggest that each type of channel can be preferentially located in one of these fractions. The determined rate constants for Ca2+ permeability through both types of channel are 0.77 +/- 0.08 min-1 (fast channels) and 0.025 +/- 0.005 min-1 (slow channels) and that for Mg2+ is 0.08 +/- 0.02 min-1. These results agree with data obtained by other groups using different experimental approaches. Dibucaine and tetracaine significantly alter the rate of Mg2+ and Ca2+ influx through the slow channels. In addition, these two local anesthetics also produce the effect that the Mg2+ influx cannot be described with only one exponential process, thus suggesting a differential effect on vesicles of different density. The increase of Ca2+ and Mg2+ permeability by dibucaine and by tetracaine is found at concentrations of these drugs that do not produce a noticeable inhibition of the (Ca2+ + Mg2+)-ATPase activity of sarcoplasmic reticulum vesicles.     

Ca2+ binding and translocation by the sarcoplasmic reticulum ATPase: Functional and structural considerations

Three experimental systems are described including sarcoplasmic reticulum (SR) vesicles, reconstituted proteoliposomes, and recombinant protein obtained by gene transfer and expression in foreign cells. It is shown that the Ca²⁺ ATPase of sarcoplasmic reticulum (SR) includes an extramembranous globular head which is connected through a stalk to a membrane bound region. Cooperative binding of two calcium ions occurs sequentially, within a channel formed by four clustered helices within the membrane bound region. Destabilization of the helical cluster is produced following enzyme phosphorylation by ATP at the catalytic site in the extramembranous region. The affinity and orientation of the Ca²⁺ binding site are thereby changed, permitting vectorial dissociation of bound Ca²⁺ against a concentration gradient. A long range linkage between phosphorylation and Ca²⁺ binding sites is provided by an intervening peptide segment that retains high homology in cation transport ATPases, and whose function is highly sensitive to mutational perturbations.     

The modification of the unidirectional calcium fluxes of sarcoplasmic reticulum vesicles by monovlent cation ionophroes

Calcium uptake by rabbit skeletal muscle sarcoplasmic reticulum vesicles in phosphate-containing media exhibits time-dependent changes that arise from changing rates of calcium influx and efflux. The monovalent cation ionophore gramicidin, added before the start of the calcium uptake reaction, delayed the spontaneous calcium release that normally occurred after approx. 6 min in such reactions; the rate of calcium efflux was inhibited while calcium influx was little affected. Under these conditions, Ca2+-activated ATPase activity could remain unaltered. Gramicidin stimulated calcium uptake irrespective of the presence of a K+ gradient across the vesicle membrane. Valinomycin stimulated calcium uptake in a manner similar to that for gramicidin even in an NaCl-containing medium lacking potassium. Thus, dissipation of a transmembrane K+ gradient is unlikely to account for the effects of these ionophores on the spontaneous changes in calcium flux rates. Addition of gramicidin to partially calcium-filled vesicles inhibited the phase of spontaneous calcium reuptake because both calcium influx and efflux wre inhibited. Addition of gramicidin to partially calcium-filled vesicles in the presence of a water-soluble protein, such as bovine serum albumin, creatine kinase or pyruvate kinase, markedly stimulated calcium uptake. This stimulatory effect was due primarily to inhibition of calcium efflux, calcium influx being minimally influenced by the ionophore. After cleavage of the 100,000 dalton ATPase to 50,000 dalton fragments, which was not associated with changes in Ca2+-activated ATPase activity or initial calcium uptake rate, gramicidin increased rather than decreased calcium content when added to vesicles after the initial maximum in calcium content. Thus, the ability of monovalent cation ionophores to block calcium efflux from calcium-filled vesicles may reflect their interaction with a portion of the Ca2+-activated ATPase protein.     

Activation of calcium transport in skeletal muscle sarcoplasmic reticulum by monovalent cations.

The rates of calcium transport and Ca2+-dependent ATP hydrolysis by rabbit skeletal muscle sarcoplasmic reticulum were stimulated by monovalent cations. The rate of decomposition of phosphoprotein intermediate of the Ca2+-dependent ATPase of sarcoplasmic reticulum was also increased by these ions to an extent that is sufficient to account for the stimulation of calcium transport and Ca2+-dependent ATPase activity. The order of effectiveness of monovalent cations tested at saturating concentrations in increasing rate of phosphoprotein decomposition is: K+, Na+ greater than Rb+, NH4+ greater than Cs+ greater than Li+, choline+, Tris+.     

Isolated bovine myocardial sarcolemma and sarcoplasmic reticulum vesicles contain tightly bound calcium-dependent protease inhibitor

Bovine myocardial sarcolemma and sarcoplasmic reticulum vesicle preparations contained calcium-dependent protease inhibitor protein. No inhibitor was detected in mitochondrial membranes. The membrane-bound inhibitor co-purified with the marker enzymes for sarcolemma and sarcoplasmic reticulum, Na+,K+-ATPase and Ca2+,K+-ATPase respectively, on isopycnic ultracentrifugation through linear sucrose density gradients. Sarcolemma and sarcoplasmic reticulum vesicles contained about 1 mg of inhibitor per g of membrane protein. However, about one-half of the inhibitor in sarcoplasmic reticulum vesicles was not tightly associated with the membrane. The membrane-bound inhibitor may function to modulate calcium-dependent proteolytic cleavage of sarcolemmal or sarcoplasmic reticulum-associated proteins.     

Kinetics of the actions of caffeine and procaine on the Ca2+-gated cation channel in sarcoplasmic reticulum vesicles

The effects of caffeine and procaine on the Ca2+-gated cation channel in sarcoplasmic reticulum (SR) vesicles were studied by measuring choline influx. The choline influx in SR vesicles was measured by following the change in light scattering intensity using a stopped flow apparatus. From the kinetic analysis of the rate of choline influx, the following results were obtained. (1) The rate of choline influx was enhanced when Ca2+ bound to the Ca2+-receptor site of the Ca2+-gated cation channel. (2) Caffeine enhanced the choline influx by increasing only the affinity of Ca2+ for the receptor site of the channel and thus regulated the equilibrium between open and closed states of the channel. The affinity increased about 14-fold upon caffeine binding. The dissociation constant of caffeine was 10 mM. (3) In contrast, procaine itself blocked the choline influx mediated by the Ca2+-gated cation channel. The blockade followed a single-site titration curve with a Ca2+-dependent dissociation constant of 0.44 mM at 2 x 10(-6) M Ca2+. The Ca2+-dependence was explained by assuming that procaine would bind to the inhibitory site only when the channel was open. (4) Procaine also inhibited the choline influx enhanced by caffeine. The blockade could be explained on the basis of the above kinetic model.     

The sodium channel from rat brain. Reconstitution of voltage-dependent scorpion toxin binding in vesicles of defined lipid composition

Purified sodium channels incorporated into phosphatidylcholine (PC) vesicles mediate neurotoxin-activated 22Na+ influx but do not bind the alpha-scorpion toxin from Leiurus quinquestriatus (LqTx) with high affinity. Addition of phosphatidylethanolamine (PE) or phosphatidylserine to the reconstitution mixture restores high affinity LqTx binding with KD = 1.9 nM for PC/PE vesicles at -90 mV and 36 degrees C in sucrose-substituted medium. Other lipids tested were markedly less effective. The binding of LqTx in vesicles of PC/PE (65:35) is sensitive to both the membrane potential formed by sodium gradients across the reconstituted vesicle membrane and the cation concentration in the extravesicular medium. Binding of LqTx is reduced 3- to 4-fold upon depolarization to 0 mV from -50 to -60 mV in experiments in which [Na+]out/[Na+]in is varied by changing [Na+]in or [Na+]out at constant extravesicular ionic strength. It is concluded that the purified sodium channel contains the receptor site for LqTx in functional form and that restoration of high affinity, voltage-dependent binding of LqTx by the purified sodium channel requires an appropriate ratio of PC to PE and/or phosphatidylserine in the vesicle membrane.     

Relation between the passive transport of calcium into vesicles of the myocardial sarcolemma and membrane potential

The effect of membrane potential on the passive 45Ca2+ uptake by cardial sarcolemmal vesicles was investigated. Membrane potentials were generated by the K+ gradient in the presence of valinomycin and were measured using fluorescent dye diS-C3-(5). It was shown that the 45Ca2+ influx into vesicles increased twice after membrane depolarization. Evaluation of the 45Ca2+ influx over a wide range of membrane potentials produced a profile similar to that of current-voltage relationships for single calcium channels in isolated cardiomyocytes. Passive 45Ca2+ transport was inhibited by 1 mM Cd2+ and Co2+. It is suggested that the voltage-dependent Ca2+ influx into vesicles occurs through Ca2+-channels.     

The effect of ionomycin on calcium fluxes in sarcoplasmic reticulum vesicles and liposomes.

Ionomycin, a recently discovered calcium ionophore, inhibits the ATP-dependent active Ca2+ transport of rabbit sarcoplasmic reticulum vesicles at concentrations as low as 10(-8) to 10(-6) M. The effect is due to an increase in the Ca2+ permeability of the membrane which is also observed on liposomes. The inhibition of Ca2+ uptake is accompanied by an increase in the Ca2+-sensitive ATPase activity of sarcoplasmic reticulum vesicles.     

Ba2+-sensitive K+ channels in luminal-membrane vesicles from pars convoluta of rabbit proximal tubule

This paper describes properties of 86Rb+ fluxes through a novel K+ channel in luminal-membrane vesicles isolated from pars convoluta of rabbit proximal tubule. The uptake of 86Rb+ into potassium salt loaded vesicles was specifically inhibited by Ba2+. The isotope accumulation is driven by an electrical diffusion potential as shown in experiments using these membrane vesicles loaded with anions of different membrane permeability and was as follows: gluconate greater than SO4(2-) greater than Cl-. Furthermore, the vesicles containing the channels show a cation selectivity with the order K+ greater than Rb+ greater than Li+ greater than Na+ = choline+.     

Ca2+ uptake and membrane potential in sarcoplasmic reticulum vesicles

The rate of calcium uptake by sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle was stimulated by inside-negative membrane potential generated by K+ gradients in the presence of valinomycin. The increase in the calcium transport rate was accompanied by a proportional increase in the rate of calcium-dependent ATP hydrolysis, without significant change in the steady state level of the phosphorylated enzyme intermediate. Changes in the sarcoplasmic reticulum membrane potential during calcium transport were monitored with the optical probe, 3,3'-diethylthiadicarbocyanine. The decrease in the absorbance of 3,3'-diethylthiadicarbocyanine at 660 nm following generation of inside-negative membrane potential was reversed during ATP-induced calcium uptake. These observations support an electrogenic mechanism for the transport of calcium by the sarcoplasmic reticulum.     

Cation transport in vesicles from secreting rabbit stomach

K+ gradient-dependent rubidium flux in vesicles obtained from stimulated rabbit stomach distinguishes two cation pathways. Selective inhibition by vanadate and the (1,2-alpha)-imidazopyridine, SCH 28080 identifies one pathway as H,K-ATPase-mediated passive cation exchange. A second pathway, additive to the first, is inhibited by the protonophore, tetrachlorosalicylanilide and is identified as a K+ conductance pathway present in these vesicles. The conductance was limited to vesicle populations obtained from the stimulated rabbit gastric mucosa and was distributed into both a light microsomal fraction and a heavier membrane fraction. 86Rb+ transport through the cation conductance exhibited a trans-stimulated cation selectivity sequence of K+ greater than Rb+ = Cs+ much greater than Li+. Potential sensitive flux was inhibited by the cyanine dye 3,3'-dipropyl-2,2'-thiodicarbo cyanine iodide, Ba2+, quinine, and the guanidinium compound 1,8-bis-guanidinium-n-octane. The presence of the conductance was correlated with K+-dependent H+ transport which did not require prolonged equilibration in K+ medium for activation. A role for the stimulus-dependent K+ conductance in gastric acid secretion could be its provision of a pathway for net K+ movement to the luminal site of the H,K-ATPase.     

The K+ channel of sarcoplasmic reticulum. A new look at Cs+ block.

K+-selective ion channels from mammalian sarcoplasmic reticulum were inserted into planar phospholipid bilayers, and single-channel currents measured in solutions containing Cs+. Current through this channel can be observed in symmetrical solutions containing only Cs+ salts. At zero voltage, the Cs+ conductance is approximately 15-fold lower than the corresponding K+ conductance. The open channel rectifies strongly in symmetrical Cs+ solutions, and the Cs+ currents are independent of Cs+ concentration in the range 18-600 mM. Biionic (Cs+/K+) reversal potentials are only 10 mV, showing that Cs+ is nearly as permeant as K+, though much less conductive. Addition of Cs+ to symmetrical K+ solutions reduces current through the channel in a voltage-dependent way. The results can be explained by a free energy profile in which the channel's selectivity filter acts in two ways: to provide binding sites for the conducting ions and to serve as a major rate-determining structure. According to this picture, the main difference between high-conductance K+ and low-conductance Cs+ is that Cs+ binds to an asymmetrically positioned site approximately 20-fold more tightly than does K+.     

Uncoupling of Ca2+ transport in sarcoplasmic reticulum as a result of labeling lipid amino groups and inhibition of Ca2+-ATPase activity by modification of lysine residues of the Ca2+-ATPase polypeptide

Limited labeling of amino groups with fluorescamine in fragmented sarcoplasmic reticulum vesicles inhibits Ca2+-ATPase activity and Ca2+ transport. Under the labeling conditions used, 80% of the label reacts with phosphatidylethanolamine and 20% with the Ca2+-ATPase polypeptide. This degree of labeling does not result in vesicular disruption or in loss of vesicular proteins and does not increase the membrane permeability to Ca2+. Fluorescamine labeling of a purified Ca2+-ATPase devoid of aminophospholipids also inhibits Ca2+-ATPase activity, suggesting that labeling of lysine residues of the enzyme polypeptide is responsible for the inhibition of Ca2+-ATPase activity in sarcoplasmic reticulum. Fluorescamine labeling interferes with phosphoenzyme formation and decomposition in both the native vesicles and the purified enzyme; addition of ATP during labeling, and with less effectiveness ADP or AMP, protects both partial reaction steps. Addition of a nonhydrolyzable ATP analog protects phosphoenzyme formation but not decomposition. The inhibition of Ca2+ transport but not of Ca2+-ATPase occurs in sarcoplasmic reticulum vesicles labeled in the presence of ATP, indicating that the transport reaction is uncoupled from the Ca2+-ATPase reaction. The inhibition of Ca2+ transport but not of Ca2+-ATPase activity is also found in sarcoplasmic reticulum vesicles in which only phosphatidylethanolamine has reacted with fluorescamine. Furthermore, the extent of labeling of phosphatidylethanolamine is correlated with the inhibition of Ca2+ transport rates. The inhibition of Ca2+ transport is a reflection of the inhibition of Ca2+ translocation and is not due to an increase in Ca2+ efflux. We propose that labeling of phosphatidylethanolamine perturbs the lipid environment around the enzyme, producing a specific defect in the Ca2+ translocation reaction.